Summary: Perturbation of the antigen (Ag) receptor by antigen or by anti-receptor antibodies induces signaling ("Signal 1") via protein tyrosine kinase activation, the phosphorylation and activation of phospholipase C gamma-1 (PLCg1), and the activation of Ras and related molecules. The activation of PLCg1, in particular, mediates phosphoinositide (PI) hydrolysis which in turn controls calcium mobilization and protein kinase C activation, obligatory events in the activation of T- or B-lymphocytes. Furthermore, certain co-stimulatory molecules (e.g., CD2, CD28) responsible for "Signal 2" affect PLCg1 activation. The proper physiologic combination of signal 1 and 2 induces immune activation, while an unbalanced signal will fail to activate cells. The laboratory is concerned with establishing the mechanisms by which immune receptors regulate PLCg1. Mutational analysis demonstrated that PLCg1 amino terminal SH2 (SH2N) domain was required and sufficient for Ag receptor-induced tyrosine phosphorylation of PLCg1 and for the association of PLCg1 with scaffold adapters. Furthermore, PLCg1 membrane translocation required the SH2N domain and was decreased by mutation of the SH3 domain. Antigen receptor induced PI hydrolysis was abrogated by mutation of either the SH2N or the carboxyl-terminal SH2 (SH2C) domain and was decreased by mutation of the SH3 domain. A role for lipid rafts, specialized membrane microdomains, as a critical compartment regulating the activation of PLCg1 was also demonstrated. In addition to coupling the Ag receptor to PLCg1-mediated PI hydrolysis, PLCg1 SH2C domain exerts additional functions, including coupling the co-stimulatory signaling pathway to downstream effectors. Hence, the SH2C domain may be at the cross road of signal 1 and 2. Our laboratory has shown that the SH2C domain acts as an auto-inhibitory domain on PLCg1 enzyme activity and that its functionality in coupling to immune receptors is regulated by a yet unknown mechanism that is likely to involve an intramolecular interaction. The cellular proto-oncogene, c-Cbl, is an adapter that associates with numerous signaling proteins, including PLCg1, involved in signal transduction by distinct receptors. C-Cbl is a major target of tyrosine phosphorylation after TCR engagement. C-Cbl binds the SH3 domain of PLCg1 and c-Cbl over-expression decreased TCR-induced PI-hydrolysis. Over-expression of c-Cbl reduced both Ag receptor-induced Ras-dependent and Ca2+-dependent transcriptional activities, suggesting that c-Cbl could act as a negative feed-back mechanism in Ag receptor-induced immune activation. In contrast, over-expression of an oncogenic c-Cbl mutant, 70Z/3 Cbl, enhanced PI-hydrolysis. These data support a model of immune receptor-induced PLCg1 activation whereby positive regulation is exerted by the scaffold-binding role of the SH2N domain and negative feed-back regulation by the SH3 domain interaction with c-Cbl. The SH2C domain, which may acts as a cross-over from signal 1 to signal 2, is regulated in a complex manner whose mechanism is the subject of ongoing studies.